Fabrega, S, P Durand, Codogno, P, Bauvy, C, Delomenie, C, Henrissat, B, Martin, BM, McKinney, C, Ginns, EI, Mornon, JP and Lehn, P (2000), "Human glucocerebrosidase: heterologous expression of active site mutants in murine null cells.", Glycobiology, 10, 11: 1217-24.
Abstract: Using bioinformatics methods, we have
previously identified Glu235 and Glu340 as the putative acid/base
catalyst and nucleophile, respectively, in the active site of human
glucocerebrosidase. Thus, we undertook site-directed mutagenesis
studies to obtain experimental evidence supporting these predictions.
Recombinant retroviruses were used to express wild-type and E235A and E340A
mutant proteins in glucocerebrosidase-deficient murine cells. In
contrast to wild-type enzyme, the mutants were found to be catalytically
inactive. We also report the results of various studies (Western
blotting, glycosylation analysis, subcellular fractionation, and confocal
microscopy) indicating that the wild-type and mutant enzymes are
identically processed and sorted to the lysosomes. Thus, enzymatic
inactivity of the mutant proteins is not the result of incorrect
folding/processing. These findings indicate that Glu235 plays a key
role in the catalytic machinery of human glucocerebrosidase and may
indeed be the acid/base catalyst. As concerns Glu340, the results both
support our computer-based predictions and confirm, at the biological
level, previous identification of Glu340 as the nucleophile by use of
active site labeling techniques. Finally, our findings may help to
better understand the molecular basis of Gaucher disease, the human
lysosomal disease resulting from deficiency in glucocerebrosidase.
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